Present therapies for HIV-1 are limited by the development of multidrug-resistant virus and by significant cumulative drug toxicities. A major goal of HIV-1 research, therefore, is the development of new classes of antiretroviral agents with novel modes of action. The HIV-1 virion infectivity factor (Vif) is an essential regulatory protein required for HIV-1 replication in primary CD4+ T-cells and macrophages. Recent landmark studies have demonstrated that Vif is a critical viral countermeasure that specifically combats an innate antiviral defense mechanism mediated by human APOBEC3G (hA3G) and related proteins. The overall goal of this project is to translate these recent discoveries into novel small-molecule Vif inhibitors that restore this important innate antiviral defense. To this end, we have developed a novel and facile gain-of-function screen for inhibitors of Vif function. Our mechanism-based assay utilizes an hA3G-luciferase (luc) reporter construct to directly monitor cellular levels of hA3G in the presence of Vif and potential Vif inhibitors. In this Phase I SBIR project, we will optimize and validate our high throughput screening assay; specific Vif inhibitors will then be identified from Progenics' diverse chemical library of over 300,000 small-molecule compounds via automated high throughput screening (HTS). "Quality hits" will be assayed for activity against HIV-1 pseudoviruses and against authentic HIV-1. Finally, potent and specific inhibitors will be characterized further in a battery of secondary assays to establish their mechanism of action and molecular target. Success in the Phase I SBIR project will be defined as the discovery of one or more candidate lead series that have the following properties: 1) potent activity in the hA3G-luc stabilization assay, 2) potent antiviral activity against a diverse panel of at least 10 HIV-1 isolates (median IC50 < 2[unreadable]M), 3) confirmed mechanism of abrogating Vif function, and 4) minimal cytotoxicity (CC50 20-fold greater than IC50). Lead candidates that exhibit this profile will be optimized for potency, specificity and bioavailability in the Phase II project to support human testing of this novel mode of HIV-1 therapy. Due to the development of multi-drug resistant virus and cumulative drug toxicities, there continues to be an urgent medical need to discover and develop new antiretrovirals. The overall goal of this proposal is to exploit new discoveries in HIV-1 research to discover small-molecule compounds that target the virion infectivity factor (Vif). Vif-specific inhibitors would have the potential to restore the body's own potent antiviral defense mechanism mediated by APOBEC3G and related proteins. [unreadable] [unreadable] [unreadable] [unreadable] [unreadable]